Matrix protein compositions for guided connective tissue growth

ABSTRACT

The present invention relates to the use of enamel matrix, enamel matrix derivatives and/or enamel matrix proteins as therapeutic and/or cosmetic agents. Said substances are sued for the manufacture of a pharmaceutical and/or cosmetic composition for actively inducing guiding and/or stimulating connective tissue growth and thus to prevent connective tissue scaring and/or contraction in a wound cavity and/or tissue defect that is characterised by a substantial loss of tissue. Comprised in the invention is in particular the use of active enamel substances for guided connective soft tissue growth and resistance to contraction in deep cavity shaped wounds following loss or removal of significant volumes of tissue, such as e.g. after surgical removal of a tumour and especially in combination with radiation therapy.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of enamel matrix, enamelmatrix derivatives and/or enamel matrix proteins as therapeutic, asprophylactic and/or as cosmetic agents. In the present invention, saidsubstances are shown to actively induce, guide and/or stimulateconnective tissue growth and to be involved in preventing connectivetissue scaring and/or contraction. Comprised in the invention is inparticular the use of active enamel substances for guided connectivesoft tissue growth and resistance to contraction in deep cavity shapedwounds following loss or removal of significant volumes of tissue, suchas e.g. after surgical removal of a tumour and especially in combinationwith radiation therapy.

BACKGROUND OF THE INVENTION

[0002] Removal of a significant volume of tissue, as is frequentlynecessary in cancer surgery, confronts the surgeon with specialtechnical and physiological problems. Removal of a significant volume oftissue often makes it difficult to close the wound properly, and whenclosing is achieved, the tissue scars and the stitches restrict themobility, which disabilitates the patient. The enclosed wounds cancollapse or fill with fluids and cell poor scar tissues. The results areoften disabling scar tissue formation, infections, pain, cosmeticanomalies or even complete loss of function of the effected body parts.For example, after surgical removal of a breast tumour, the contractionof the wound cavity often leads to shrinkage of the breast that by farexceeds the volume of the originally removed tissue. Furthermore,adjuvant radiation therapy increases the above mentioned problemsdrastically. Today, these post-surgical conditions are treated bysilicon inlays, secondary palliative surgery, pain and infectioncontrolling drugs, or in the worst case by amputation. Needles to say,these conditions and measurements all cause severe problems for thepatients, physically, esthetical and emotionally.

[0003] Enamel matrix proteins, present in the enamel matrix, are mostwell-known as precursors to enamel. Prior to cementum formation, enamelmatrix proteins are deposited on the root surface at the apical end ofthe developing tooth-root. There is evidence that the deposited enamelmatrix is the initiating factor for the formation of cementum. Again,the formation of cementum in itself is associated with the developmentof the periodontal ligament and the alveolar bone. As shown by thepresent inventors prior to the present invention, enamel matrix proteinscan therefore promote periodontal regeneration through mimicking thenatural attachment development in the tooth (Gestrelius S, LyngstadaasSP, Hammarstrøm L. Emdogain—periodontal regeneration based onbiomimicry. Clin Oral Invest 4:120-125 (2000).

[0004] The enamel matrix is composed of a number of proteins, such asamelogenin, enamelin, tuft protein, proteases, and albumin. Amelogenins,the major constituent of the enamel matrix, are a family of hydrophobicproteins derived from a single gene by alternative splicing andcontrolled post secretory processing. They are highly conservedthroughout vertebrate evolution and demonstrate a high overall level ofsequence homology among all higher vertebrates examined (>80%). In fact,the sequences of porcine and human amelogenin gene transcript differonly in 4% of the bases. Thus, enamel matrix proteins, although ofporcine origin, are considered “self” when encountered in the human bodyand can promote dental regeneration in humans without triggeringallergic responses or other undesirable reactions.

[0005] Enamel matrix derivative (EMD), in the form of a purified acidextract of proteins from pig enamel matrix has previously beensuccessfully employed to restore functional periodontal ligament,cementum and alveolar bone in patients with severe tooth attachment loss(Hammarström et al., 1997, Journal of Clinical Periodontology 24,658-668).

[0006] In studies on cultured periodontal ligament cells (PDL), it wasfurthermore shown that the attachment rate, growth and metabolism ofthese cells were significantly increased when EMD was present in thecultures. Also, cells exposed to EMD showed increased intracellular cAMPsignalling and autocrine production of growth factors, when compared tocontrols. Epithelial cells on the other hand, increased cAMP signallingand growth factor secretion when EMD was present, but theirproliferation and growth were inhibited (Lyngstadaas et al., 2001,Journal of Clinical Periodontology 28, 181-188).

[0007] Enamel proteins and enamel matrix derivatives have previouslybeen described in the patent literature to be able to induce hard tissueformation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)),binding between hard tissues (EP-B-0 337 967 and EP-B0 263 086) and openwound healing, such as of skin and mucosa (WO 9943344).

[0008] The present application relates to the beneficial effects of EMDon guided connective soft tissue growth and resistance to contraction inclosed wounds following loss or removal of significant volumes oftissue, such as e.g. after tumour surgery and especially in combinationwith radiation therapy, effects that are both unexpected and surprising.

DISCLOSURE OF THE INVENTION

[0009] Maintenance, repair and regeneration of differentiated tissue isguided by several tissue specific growth factors. However, so far allattempts to apply individual growth factors to regeneration of humantissue in therapy have failed in clinical trials. This is thought to bedue to growth factors being pluripotent signal factors that work inconcert. In an intricate teamwork, they induce and modulate tissuegrowth, differentiation and maturation during development andorchestrate healing, repair and regeneration of diseased tissue. Theenamel matrix derivatives (EMD) of the present invention, however, areable to circumvent this problem, as they can induce not only one but anorchestrated cascade of factors naturally found in tissues developingadjacent to the enamel matrix. They mimic the natural environment of adeveloping tissue and thus mimic a natural stimulation for tissueregeneration, cell differentiation and/or maturation.

[0010] The present invention is based on the surprising finding thatenamel matrix, enamel matrix derivatives and/or enamel matrix proteins(the term “an active enamel substance” is in the following also used foran enamel matrix, an enamel matrix derivative or an enamel matrixprotein) do not only promote periodontal ligament cell growth, but alsostimulate non-periodontal fibroblast cell growth and differentiation,whereas epithelial cell growth or differentiation is not stimulated bythe presence of active enamel substances. The use of a pharmaceutical orcosmetical composition comprising an active enamel substance, asdescribed in the present invention, thus relates to the selectivestimulation of mesodermal and/or endodermal cell growth, includinggrowth of cartilage, bone and connective tissue, striated and smoothmuscles, the heart, blood and lymph vessels and cells, the kidneys,gonades (ovaries and testes), the genital ducts, serous membranes liningthe body cavities (pericardial, pleural and peritoneal), the spleen, thecortex of the suprarenal gland, the epithelial lining of thegastrointestinal and respiratory tracts, the parenchyma of the tonsils,thyroid gland, parathyroid glands, thymus, liver and pancreas, theepithelial lining of the urinary bladder and most of the ureta, theepithelial lining of the tympanic cavity, tympanic antrum and auditorytube, as opposed to ectodermal cell growth including growth of thecentral or peripheral nervous system, the epidermis and its appendages(hair and nails), the mammary glands, pituitary gland, the subcutaneousglands and the enamel of teeth, which is not stimulated by theapplication of said composition comprising an active enamel substance.

[0011] As shown by the present inventors (see experiment 1), theincreased attachment rate of non-periodontal fibroblast cells that growon active enamel substances demonstrates that an enamel protein basedmatrix mimics an extracellular matrix. This mimicry facilitates rapidattachment of these cells. The observed rise in growth rate andmetabolism in these fibroblast cells, growing on active enamelsubstances, further proves that active enamel substances provide anextracellular matrix that stimulates fibroblast cells to speed up theirmetabolism. Also, a rise in growth rate is reflected in the increase ofDNA synthesis, indicating that cell proliferation is up-regulated inthese cultures. Furthermore, since the increase in utilisation of[³⁵S]-methionine in these fibroblast cells exceeds the rise in growthrate, some of the added metabolic activity also reflects a boostedanabolism and/or secretion of extracellular proteins.

[0012] The surprising findings described above lead to the envisioningof substantially new possibilities for the use of active enamelsubstances. One embodiment, described in the present invention,comprises the use of active enamel substances for preparing apharmaceutical or cosmetical composition that is to be used as a fill-inapplication for significant tissue loss wounds that would otherwise leadto painful and/or disfiguring scarring of a mammal's body. Thesuccessful application of said composition, as described herein, leadsto increased neogenesis and tissue-specific gain of soft tissue, muscle,blood and lymph vessels, tendons, and cartilage.

[0013] Not intended to be within the scope of the present invention isthe treatment of shallow open wounds in soft tissue with active enamelsubstances.

[0014] A preferred embodiment of the present invention relates to theuse of an active enamel substance for the preparation of apharmaceutical or cosmetic composition that is used as a beneficialagent for the enhancement or improvement of guided connective tissuegrowth into soft tissue defects following significant tissue loss due totrauma, infectious diseases, necrosis, removal of neoplasms or othercytoreductive surgical interventions. As demonstrated in theexperimental section herein, the active enamel substance exertsespecially useful effects in guiding and stimulating connective tissuegrowth into a significant tissue loss following the surgical removal ofa tumour. Further, another preferred embodiment relates to the use ofsaid active enamel substance for the preparation of a pharmaceutical orcosmetic composition for guiding and stimulating connective tissuegrowth following post surgical tumour treatment after significant tissueloss, such as after radiation therapy.

[0015] The present invention thus provides optimal means to refill aloss of tissue that is left from a surgically removed tissue, such ase.g. a tumour, with new fibroblasts that are stimulated to proliferatein the wound cavity. To this means, the cavity is filled with apreparation of active enamel substances, such as e.g. with EMDOGAIN®(BIORA AB, Sweden), which will successively be degraded and replaced bynew fibroblasts. Thereby, the cavity will be protected from collapsingand the surrounding tissue will be protected from contracting, andfinally, the new grown soft tissue will act as a natural replacement forthe surgically removed tissue. Furthermore, the unique property ofactive enamel substances to stimulate proliferation and differentiationof selective tissue types, will in the above mentioned embodiment onlystimulate the neogenesis of mesodermal or endodermal cells, but notstimulate any ectodermal cell growth.

[0016] In an especially preferred embodiment of the invention, an activeenamel substances can therefore be used for the preparation of apharmaceutical or cosmetic composition for filling of a cavity woundthat is characterised by substantial loss of tissue after surgicalremoval of a breast tumour. Breast tumours are mainly glandular tumours,i.e. that active enamel substances will not stimulate the regrowth ofthe tumour tissue, glands being of ectodermal origin, but only stimulatethe fill-in of the cavity with new connective tissue. In a laterscenario, a person skilled in the art will have no difficulties tofurther envision the stimulation of regrowth of blood vessels andinnervation that will follow the fibrotic fill-in of the wound.

[0017] The use of a composition comprising an active enamel substance,as described in the present invention, will prevent contraction ofconnective tissue, following significant tissue loss due to surgicaltumour treatment such as surgical removal of significant tumour tissueand post surgical tumour treatment, such as, for example but notexclusively, radiation therapy. The use of active enamel substances inaccordance to this invention in a tension-free closure may be associatedwith less pain and less incidence of postoperative fluid accumulation(seroma). Furthermore, compositions comprising an active enamelsubstance will also help increase tensile strength of the wound and thusbe beneficial for improving yet another aspect of the repair process.

[0018] Accordingly, the invention relates to the use of an active enamelsubstance for the preparation of a pharmaceutical or cosmeticcomposition i) for improving connective tissue fill of a significanttissue loss and/or defect, and/or ii) for avoiding soft tissuecontraction following significant tissue loss due to e.g. tumour removaland thus limiting post surgical therapy complications.

LEGENDS TO FIGURES

[0019]FIG. 1: Normal Human Dermal Fibroblast (NHDF) cell attachment rateduring the first hours after seeding is nearly five times more efficientwhen the surface of the culture dish is coated with EMD. n=6 error barsgive ±SD

[0020]FIG. 2: Density plot of cultured NHDF cells show that cellsgrowing in presence of EMD proliferate faster than controls. Fivedifferent areas in each culture were counted in each of six parallelcultures at each time point (n=30), error bars are ±SD.

[0021]FIG. 3: Utilization of [³⁵S]methionine pulse plotted against daysin NHDF cultures after seeding. The presence of EMD in the culturessignificantly increases the metabolic rate of NHDF cells. n=9, errorbars give ±SD.

[0022]FIG. 4: DNA synthesis analyzed by BrdU incorporation during DNAreplication show that NHDF cells significantly increase synthesis ofnucleic acids in presence of EMD. n=6, error bars give ±SD.

[0023]FIG. 5: Radiated cells growing in the presence of EMD increasetheir number twice as fast as unstimulated cultures (FIGS. 5A and 5B)

DETAILED DISCLOSURE

[0024] The present invention relates to the use of enamel matrix, enamelmatrix derivatives and/or enamel matrix proteins for actively inducing,guiding and/or stimulating connective tissue growth and for beinginvolved in preventing connective tissue scaring and/or contraction indeep cavity wound healing.

[0025] The majority of tissue cavities and defects are filled throughreparative processes, i.e. the new tissue that is formed (scar tissue)is often of different volume and structurally and chemically unlike theoriginal tissue. In the early stage of the tissue repair, one process isalmost always involved, the formation of a transient connective tissuein the area of the tissue injury. This process starts by forming of anew extracellular collagen matrix by fibroblasts. Said new extracellularcollagen matrix supports the connective tissue during the final healingprocess. However, in defects wherein a significant volume of tissue hasbeen removed or lost, the defect or deep tissue-cavity will not fillwith connective tissue and extracellular collagen based matrix, but asignificant part of the defect volume will fill with cell free exudatefrom the surrounding tissue. To allow for connective tissue fill of thewhole cavity, the exudate has to be drained out so that matrix formationand subsequent connective tissue growth can occupy most of the cavity.

[0026] Under normal circumstances, the body provides mechanisms forhealing tissue cavities in order to restore the function and integrityof the involved tissue or body part. However, the recovering time can bevery long and the defect may persist for an extended period of time,i.e. months or even years. During this time, the patient is oftendisabled and suffers from pain, discomfort and complications that needregular professional attendance.

[0027] The repair of tissue defects and/or cavities follows theclassical wound healing stages that normally include inflammation(normally 1-3 days), migration (normally 1-6 days), proliferation(normally 3-24 days) and maturation (normally 1-12 months). The healingprocess is a complex and well-orchestrated physiological process thatinvolves migration, proliferation and differentiation of a variety ofcell types as well as synthesis of matrix components.

[0028] The healing process may be separated into the following threephases:

[0029] i) Haemostasis and Inflammation

[0030] When platelets are present outside the circulatory system andexposed to thrombin and collagen, they become activated and theyaggregate. Thus, platelets Initiate the repair process by aggregatingand forming a temporary plug to ensure haemostasis and prevent invasionfrom bacteria. The activated platelets initiate the coagulation systemand release growth factors like platelet-derived growth factor (PDGF)and epidermal growth factors (EGFs) and transforming growth factors(TGFs).

[0031] The first cells to invade the wound area are neutrophils followedby monocytes which are activated by macrophages.

[0032] The major role of neutrophils appears to be clearing the wound ofcontaminating bacteria or defending the wound against contaminatingbacteria and to improve the healing of the wound by removing dead cellsand platelets. The infiltration of neutrophils ceases within about thefirst 48 hours, provided that no bacterial contamination is present inthe wound. Excess neutrophils are phagocytosed by tissue macrophagesrecruited from the circulating pool of blood-borne monocytes.Macrophages are believed to be essential for efficient wound healing inthat they are also responsible for phagocytosis of pathogenic organismsand a clearing up of tissue debris. Furthermore, they release numerousfactors involved in subsequent events of the healing process. Themacrophages attract fibroblasts that start the production of collagen.

[0033] ii) Granulation Tissue Formation

[0034] Within 48 hours after wounding, fibroblasts begin to proliferateand migrate into the wound space from the connective tissue at the woundedge. The fibroblasts produce collagens and glycosaminoglycans and interalia low oxygen tension at the wound stimulates proliferation ofendothelial cells. The endothelial cells give rise to the formation of anew capillary network. At this stage, the wound area, i.e. the cavity,is further decreased by contraction. In the case of deep, cavity-like,soft tissue enclosed defects, e.g. like those present following surgicalremoval of a breast tumour, this contraction affects both the appearanceof the body part as well as the performance, and often causes pain anddiscomfort.

[0035] At this stage, if necessary as adjuvant therapy after removal ofmalignant tumours, radiation therapy is applied. Said therapy aims atremoving residual cancer cells and local metastases from the tissue orbody part that has been treated. Besides killing all proliferatingcancer cells, this therapy also affects all normal cells that areundergoing mitosis, and imposes structural damage to DNA and proteins inthe repairing tissue. The effect of this damage is a severe slow down ofthe cellular repair processes, while the extracellular processes remainmostly unaffected. With time, this induced imbalance between cellulargrowth and extracellular processes causes severe contractions of thewound surfaces and thus the cavity and/or defect. These contractions areoften so painful and disfiguring that the tissue or body part inquestion is removed and substituted by prosthesis where possible, e.g.removal of major part of the breast and reconstruction by silicon inlay.In the cases where removal is not compatible with life, reconstructivesurgery is the only option. However, because of the radiation damage tothe DNA, the tissue does not have the ability to conduct the normalrepair sequence and the chances are that the new surgical defect alsoundergoes contraction with subsequent need for even more reconstructivesurgery.

[0036] iii) Tissue Remodelling

[0037] As soon as the defect is completely filled with scar tissue,remodelling of the tissue begins. During this phase the scar tissue issubstituted with a more organised type of tissue that aims at restoringthe strength, function, performance and appearance of the tissue/bodypart in question. This phase typically lasts for several years after theinitial defect. Also this stage is severely hampered by radiationtherapy that causes contraction and scarring.

[0038] All of the above-mentioned processes take considerable time. Therate of healing is influenced by the wound's freedom from infection, thegeneral health of the individual, presence of foreign bodies, etc. Somepathologic conditions like infection, maceration, drying out, generallypoor health and malnutrition can, if left untreated for a longer time,lead to formation of a chronically inflamed or infected tissue defect orcavity that is very difficult to cure. Furthermore, since the primarydefect is caused by removal of significant volumes of tissue, surgicalremoval of the defunct tissue is not desirable or even possible. Theseconditions therefore severely affect the quality of life of a patientand can also be extremely disabling or even life threatening.

[0039] Traditionally, cavity like, soft tissue defects have been treatedby closing them with sutures with a draining device present that allowswound exudate and puss to escape from the cavity during the initialphases of the healing process. While favourable for cellular filling ofthe defect, this strategy delays the healing because a foreign body isleft in the wound that can provoke inflammation and makes a gateway forinvading micro flora. Delayed wound healing or inflammation canexacerbate fibrosis and subsequent tissue contraction.

[0040] Until the tissue defect is filled with cellular connectivetissue, the defect remains at risk of continued or new infection,inflammation and/or severe contraction. Therefore, the quicker thedefect can heal, the sooner the risk is removed. Thus, the use of activeenamel substances according to the present invention represent meansthat can influence the rate of connective tissue filling andorganisation and favourably influence the healing capacity of radiatedand/or non-radiated connective tissues. Furthermore, as almost alltissue repair processes include the early connective tissue formation, astimulation of this and the subsequent processes are also contemplatedto improve the quality and quantity of tissue defect filling.

[0041] In the present context, the term “clinical healing” is used todenote a situation wherein no tissue interruption can be visuallyobserved and only discrete signs of inflammation are present, such as alight redness or a discretely swollen tissue. In addition, no complaintsof pain are present when the organ is relaxed or untouched.

[0042] As mentioned above, the invention relates to the use of enamelmatrix, enamel matrix derivatives and/or enamel matrix proteins as anagent for stimulation of connective tissue growth, i.e. as an agent thataccelerates, stimulates and/or promotes the growth of connective tissuecells. Accordingly, an important aspect is the use of an active enamelsubstance as a repair agent to prevent wound contraction, both surgicaland radiation induced. Furthermore, secondarily to the biologicaleffect, the administration of active enamel substances will render painrelief, because of a more rapid filling process of the defect and/ortissue-cavity and less wound contraction.

[0043] The enamel matrix, enamel matrix derivatives and/or enamel matrixproteins may be applied either directly into the soft tissue defectprior to suturing or it may be injected into the wound cavity aftersuturing. The volume/amount of enamel matrix, enamel matrix derivativesand/or enamel matrix proteins applied will differ from case to case andtissue to tissue, but generally, the therapy will aim at replacing thevolume of the lost tissue. However, in cases where an increase ordecrease of the volume of a tissue or body part, e.g. a breast, isdesirable, the enamel matrix, enamel matrix derivatives and/or enamelmatrix proteins may be applied in surplus or deficit to acquire thedesired outcome. The active enamel substance may be used as such, or maybe used in a suitable preparation or pharmaceutical composition.

[0044] The present inventors have in experiments with cultured dermalfibroblasts shown that cells that have received therapeutic dosages ofionising radiation can be stimulated with EMDOGAIN® (BIORA AB, Sweden)to replicate and grow almost at the speed of normal untreated cells. Theability of radiated cells to recover from the damage and to restorenormal functions is crucial if contraction complications are to beavoided following cancer surgery and radiation therapy. Thus, theobservation that active enamel substances can induce such beneficialchanges in radiated fibroblasts proves the concept of the presentinvention.

[0045] Moreover, the inventors have found that active enamel substanceshave the ability to stimulate fibroblast invasion, proliferation andgrowth. Furthermore, there are indications that the application ofactive enamel substances leads to improved defect fill that reduces postoperation and post radiation contractions in the defect. Also, theinventors have observed that the inflammation stage is shortened and thetypical signs such as warmth, redness, oedema and pain are lessnoticeable, and that new tissue is formed more rapidly after applicationof active enamel substances. Thus, the observed time for wound healing(e.g. after surgery) is significantly shortened as compared to surgerywithout the use of active enamel substances.

[0046] Interestingly, the inventors have also observed that various cellcultures of fibroblasts (embryonic, dermal, derived from the periodontalligament, fish fin or bird skin), produce twice as much transforminggrowth factor (TGF-β1) when stimulated with EMDOGAIN® (BIORA AB,SWEDEN), compared to non-stimulated cultures. As TGF-β1 is considered tobe of central importance in the neogenesis and reorganisation ofconnective tissue, these findings further support the concept of thepresent invention.

[0047] Significant tissue loss or defect following trauma orcytoreductive surgery normally produces cavities at the location of theremoved tissue. These cavities usually fill with fluid and/or fibrous,dense scar tissue, causing the function and appearance of the remainingtissue to be severely impaired. If normal connective tissue growth isinduced to fill such cavities, it dramatically improves the functionaland esthetical outcome of cytoreductive surgery and trauma treatment.

[0048] The preparation of an active enamel substance according to thepresent invention is effective for treating a wide variety of differenttypes and sizes of wounds. Regeneration of experimentally provokedperiodontal wounds have previously been described by the inventors andis not intended to be within the scope of the present invention, neitheris healing of wounds in skin or mucous membranes.

[0049] In the present context the terms “wound cavity”, “tissue cavity”and “tissue defects” denote an internal bodily injury with disruption ofthe normal integrity of tissue structures that follows from a procedureand/or trauma that involves removal and/or loss of a significant amountof tissue from the body or a body part. The term is also intended toencompass the terms “surgical lesions”, “necrosis”, “abscesses”,“mucocele”, “cysts”, “fistulas”, “excavations”, “alveola” and all otherterms used to describe abnormal cavities within the human body ortissue.

[0050] A deep cavity-shaped wound is in the present context clearlydefined in contrast to a shallow, open wound on the surface of a tissue,especially on the epidermis of a body, such as an abrasive-wound or amucosal wound. A cavity-shaped wound creates a deep, substantiallyhollow space in the injured tissue and will either fill with fluids,scar tissue and/or collapse in the natural repair process.

[0051] Another way of classifying wound cavities or tissue defects is asi) significant tissue loss due to surgical incisions' radiationtreatment, abrasions, lacerations, burns (chemical and thermal), donorsite wounds and substantial bites, or as ii) significant tissue loss dueto pathological conditions including ischemic ulcers, fistulae,neoplasms, tumours, hammartomas, infections, necrosis, and infarctions.

[0052] A “significant tissue loss” in the context of the presentinvention is meant to comprise an amount of tissue that is typicallyremoved from an original tissue, for example but not exclusively, due totrauma or due to surgery, and that results in a deep wound in saidtissue and/or that impairs function and/or appearance of said body part.A “significant tissue loss” in the context of the present invention willultimately lead to scarring.

[0053] In one embodiment of the invention, significant tissue losscomprises e.g. the loss of at least 5% of weight of tissue, compared tothe original weight of an organ or body part. Such a loss will in thiscontext comprise a reduction of tissue mass of between 5% and 75%, suchas between at least 5%, 5.5%, 60/%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or at least between 20%and 75%. In another embodiment of the present invention, the tissue losscomprises a reduction of tissue weight of at least 25%, such as at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85%.

[0054] In an especially preferred embodiment of this invention, the useof an active enamel substance for the preparation of a pharmaceutical orcosmetic composition for filling a tissue cavity and/or defect that ischaracterised by a substantial tissue loss is comprised, wherein thetissue loss is due to the surgical removal of a tumour and/or postsurgical treatment with radiation therapy. In this specific embodiment,“significant tissue loss” in the context of the present invention ismeant to comprise an amount of tissue ranging between about 1 ml-250 ml.Such a range will thus include an amount of tissue of about 1 ml, 1.25ml, 1.5 ml, 2 ml, 2.25 ml, 2.5 ml, 2.75 ml, 3 ml, 3.25 ml, 3.5 ml, 3.75ml, 4 ml, 4.25 ml, 4.5 ml, 4.75 ml, 5 ml, 5.25 ml, 5.5 ml, 5.75 ml, 6ml, 6.25 ml, 6.5 ml, 6.75 ml, 7 ml, 7.25 ml, 7.5 ml, 7.75 ml, 8 ml, 8.25ml, 8.5 ml, 8.75 ml, 9 ml, 9.25 ml, 9.5 ml, 9.75 ml, 10 ml, −15 ml, 20ml, 25 ml, 50 ml, 75 ml, 100 ml, 150 ml, 200 ml or 250 ml.

[0055] In certain embodiments, wherein the tissue loss is e.g. due tothe surgical removal of the majority of an organ or body part and/or alarge tumour, the amount of tissue will of course include even largervolumes than 250 ml, such as between at least 250 ml and 500 ml,depending on the original size of the organ or body part and/or tumour.

[0056] The kinds of tissue cavities to be treated according to theinvention include diverse orbital cavity or periorbital soft tissue andbony defects due to trauma, removal of benign or malignant neoplasms,tumours of the head and neck, abdomen and/or the extremities,particularly ovarian cancer and/or prostate cancer. They furthercomprise closed abdominal wounds, cavity wounds with negative pressure,penetrating thoracic and abdominal trauma wounds and/or abdominalgunshot injuries.

[0057] In the present context, a “tumour” stands for any new-growth oftissue in which the multiplication of cells is uncontrolled andprogressive. The term “tumour” is herein equivalent to “neoplasm”.

[0058] The invention comprises the use of an active enamel substance forthe preparation of a pharmaceutical or cosmetic composition for fillinga wound cavity and/or tissue defect that is due to surgical removal ofprimary and/or metastatic solid tumours and carcinomas of the breast,colon, rectum, lung, oropharynx, hypopharynx, oesophagus, stomach,pancreas, liver, gallbladder, bile duct, small intestine, urinary tractincluding kidney, bladder and urothelium, female genital tract includingcervix, uterus, ovaries, choriocarcinoma and gestational trophoblasticdisease, male genital tract including prostate, seminal vesicles, testesand germ cell tumours, endocrine glands including thyroid, adrenal, andpituitary, skin including hemangiomas, melanomas, sarcomas arising frombone or soft tissues and Kaposi's sarcoma, tumours of the head, nerves,eyes, and meninges including astrocytomas, gliomas, glioblastomas,retinoblastomas, neuromas, neuroblastomas, Shwannomas and meningiomas,solid tumours arising from hematopoietic malignancies such as leukemiasand including chloromas, plasmacytomas, placques and tumours of mycosisfungoides and cutaneous T-cell lymphoma/leukemia and/or lymphomasincluding both Hodgkin's and non-Hodgkin's lymphoma's.

[0059] In one embodiment of the present invention, tissue removed byresection during surgery includes not only tissue suspected by thesurgeon of being neoplastic, but also includes an amount of healthytissue taken because the precise tumour margins can not be ascertainedby the surgeon. Coupled with the devastating risk of not removingneoplastic tissue resulting in tumour recurrence, surgical protocoldictates that healthy tissue is taken in order to ensure the removal ofneoplastic tissue. Of course, the final determination as to whether theresected tissue is malignant falls to the pathologist who receives thetissue removed during the surgical procedure.

[0060] The present invention further relates to the use of an activeenamel substance for the preparation of a pharmaceutical composition fora reconstruction that can be a post-mastectomy procedure, apost-traumatic procedure, or a procedure done to enlarge or decrease thevolume of a breast. A reconstruction can be contemporaneous with amastectomy or can be delayed, taking place over one or morepost-mastectomy surgical procedures. In accordance with the invention, adelayed procedure comprises: a multistage procedure where a mastectomyis performed with contemporaneous placement of an expander, and asubsequent procedure when the reconstruction is performed; a mastectomy;a subsequent procedure when an expander is placed, and a subsequentprocedure when reconstruction is performed; revisions to a previousreconstruction; or, the placing or modifying of breast implant materialsthat comprise a pharmaceutical composition according to the presentinvention.

[0061] Thus, the invention provides means for total and/or partialbreast reconstruction that is either delayed or immediate. With animmediate reconstruction, the patient does not experience a mastectomydeformity and accompanying emotional trauma; however, for many women adelayed reconstruction is medically indicated. For patients who haveundergone a standard modified radical mastectomy, delayed autologousreconstruction is accomplished after expansion of the skin envelope.

[0062] By use of preferred embodiments, a breast reconstructionaccording to the present invention can be performed on both breasts.Thus, if a subsequent breast cancer occurs in the contralateral breast,the same procedure can be performed. Alternatively, if a bilateralbreast cancer is present, one or both breasts can be reconstructed withthis technique.

[0063] As used herein, a “substantially circumareolar incision”comprises an incision that circumscribes at the perimeter of thenipple-areolar complex; in instances where a breast reduction and/or anipple-areolar repositioning is to be performed, an incision thatcircumscribes at the perimeter of the nipple-areolar complex andincludes additional breast skin; and, an incision that approximates theareolar perimeter yet is within the area of the areola. Preferably,particularly for immediate embodiments of the invention, thecircumareolar incision closely corresponds to the perimeter of theareola, and is at or within the margin of the areola. A substantiallycircumareolar incision also comprises radial or wedge skin incisions atthe border of the areola. Radial or wedge skin incisions at the borderof the areola are less ideal since they create scarring that does notcorrespond to a natural tissue plane.

[0064] Advantageously, a reconstructed breast produced in accordancewith the present invention has better contour and projection thanreconstructed breasts that resulted from procedures well known to theskilled artisan.

[0065] Preferred embodiments of the procedure of the present inventionare performed following a circumareolar mastectomy. A circumareolarmastectomy eliminates the transverse mastectomy scar that was aconsequence of prior reconstruction procedures. To make mostadvantageous use of a circumareolar mastectomy, it is preferred that thesurgeon limit any biopsy and subsequent mastectomy skin excision to theregion of the nipple-areolar complex. When the skin excision in amastectomy is limited to the region of the nipple-areolar complex, theskin envelope of the breast is completely preserved. Since the biopsyincision is generally removed at the time of the mastectomy, biopsyincisions outside the region of the areola often necessitate anon-preferably large skin incision on the breast skin envelope. With alarge skin incision, the mastectomy scar is not camouflaged at theborder of the areola, and the reconstructed breast is less likely tohave a normal contour.

[0066] A mastectomy excision through a substantially circumareolarincision that is larger than the areolar perimeter can be performed inaccordance with the invention, particularly for patients for whom abreast reduction and/or nipple-areolar repositioning is indicated; forsuch patients, a “substantially circumareolar incision” comprises anincision that corresponds to the perimeter of the areola and comprisesan incision that corresponds to a standard pattern reduction orrepositioning incision. In general, if a skin resection extends beyondthe perimeter of the nipple-areolar complex, the resulting mastectomyscar is more readily apparent.

[0067] Alternatively, yet also in accordance with the present invention,a variable amount of areola may be left with the breast skin.

[0068] Radiation therapy is known to those skilled in the art to be lessdistorting if performed after a breast reconstruction. Patientsnecessitating postoperative radiation therapy will be candidates for theuse of the present invention. The use will alter the patient'spostoperative prognosis favourably. Size or stage of the breast cancerwill not limit applicability of the procedure. If a modified radicalmastectomy with a large elliptical skin excision is indicated, a delayedreconstruction in accordance with the invention is generally used.

[0069] Additionally to the use in breast surgery, the present inventionfurther relates to the use of an active enamel substance for thepreparation of a pharmaceutical composition for reconstruction or forguided connective tissue growth after vaginal, urinal and/or analsurgery or surgery on urine bladder, womb or intestines.

[0070] In the scope of the present invention are also other uses forrepairing soft tissue defects, such as soft tissue defects resultingfrom incisional hernias and soft tissue defects resulting fromextirpative tumour surgery. Other applications of the present inventioninclude laparoscopic inguinal hernia repair, standard inguinal herniarepair, umbilical hernia repair, paracolostomy hernia repair, femorahernia repair, lumbar hernia repair, and the repair of other abdominalwall defects, thoracic wall defects and diaphragmatic hernias anddefects.

[0071] In yet another embodiment, the present invention relates to theuse of an active enamel substance for the preparation of apharmaceutical or cosmetic composition for the enlargement of a breastor any other soft tissue of a mammal, wherein a certain amount of saidcomposition, is inserted into a mammalian tissue without any prior lossof tissue. This embodiment incorporates the use of an active enamelsubstance for purely cosmetic reasons and is not limited to apost-surgical treatment of a tumour, such as e.g. breast cancer. In thiscontext, the scope of the invention therefore relates to cosmeticmethods for treating a human being with an active enamel substance forstimulating the neogenesis of soft tissue, characterised by inserting orinjecting a suitable pharmaceutical composition comprising active enamelsubstances, such as e.g. EMDOGAIN® (BIORA AB, Sweden) into the organand/or body part, wherein cosmetical enlargement or filling is desired.

[0072] Enamel matrix is a precursor to enamel and may be obtained fromany relevant natural source, i.e. a mammal in which teeth are underdevelopment. A suitable source is developing teeth from slaughteredanimals such as, e.g., calves, pigs or lambs. Another source is e.g.fish skin.

[0073] Enamel matrix can be prepared from developing teeth as describedpreviously (EP-B-0 337 967 and EP-B-0 263 086). The enamel matrix isscraped off and enamel matrix derivatives are prepared, e.g. byextraction with aqueous solution such as a buffer, a dilute acid or baseor a water/solvent mixture, followed by size exclusion, desalting orother purification steps, followed by freeze-drying. Enzymes mayalternatively be deactivated by treatment with heat or solvents, inwhich case the derivatives may be stored in liquid form withoutfreeze-drying.

[0074] As an alternative source of the enamel matrix derivatives orproteins one may also use generally applicable synthetic routes, wellknown to a person skilled in the art, or use cultivated eukaryoticand/or prokaryotic cells modified by DNA-techniques. The enamel matrixproteins may thus be of recombinant origin and alternatively geneticallymodified (see, e.g., Sambrook, 3. et al.: Molecular Cloning, Cold SpringHarbor Laboratory Press, 1989).

[0075] In the present context, enamel matrix derivatives are derivativesof enamel matrix which include one or several enamel matrix proteins orparts of such proteins, produced naturally by alternate splicing orprocessing, or by either enzymatic or chemical cleavage of a naturallength protein, or by synthesis of polypeptides in vitro or in vivo(recombinant DNA methods or cultivation of diploid cells). Enamel matrixprotein derivatives also include enamel matrix related polypeptides orproteins. The polypeptides or proteins may be bound to a suitablebiodegradable carrier molecule, such as polyamine acids orpolysaccharides, or combinations thereof. Furthermore, the term enamelmatrix derivatives also encompass synthetic analogous substances.

[0076] Proteins are biological macromolecules constituted by amino acidresidues linked together by peptide bonds. Proteins, as linear polymersof amino acids, are also called polypeptides. Typically, proteins have50-800 amino acid residues and hence have molecular weights in the rangeof from about 6,000 to about several hundred thousand Dalton or more.Small proteins are called peptides or oligopeptides.

[0077] Enamel matrix proteins are proteins that normally are present inenamel matrix, i.e. the precursor for enamel (Ten Cate: Oral Histology,1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1:282-91),or proteins which can be obtained by cleavage of such proteins. Ingeneral, such proteins have a molecular weight below 120,000 Dalton andinclude amelogenins, non-amelogenins, proline-rich non-amelogenins andtuftelins.

[0078] Examples of proteins for use according to the invention areamelogenins, proline-rich non-amelogenins, tuftelin, tuft proteins,serum proteins, salivary proteins, ameloblastin, sheathlin, andderivatives thereof, and mixtures thereof. A preparation containing anactive enamel substance for use according to the invention may alsocontain at least two of the aforementioned proteinaceous substances.Moreover, other proteins for use according to the invention are found inthe marketed product EMDOGAIN®) (BIORA AB, Sweden).

[0079] EMDOGAIN® (BIORA AB, S-205 12 Malmö, Sweden) contains 30 mgEnamel Matrix protein, heated for 3 hours at about 80° C. in order toinactivate residual proteases, and 1 ml Vehicle Solution (PropyleneGlycol Alginate), which are mixed prior to application, unless theprotein and the Vehicle are tested separately. The weight ratio is about80/8/12 between the main protein peaks at 20, 14 and 5 kDa,respectively.

[0080] In general, the major proteins of an enamel matrix are known asamelogenins. They constitute about 90% w/w of the matrix proteins. Theremaining 10% w/w includes proline-rich non-amelogenins, tuftelin, tuftproteins, serum proteins and at least one salivary protein; however,other proteins may also be present such as, e.g., amelin (ameloblastin,sheathlin) which have been identified in association with enamel matrix.Furthermore, the various proteins may be synthesised and/or processed inseveral different sizes (i.e. different molecular weights). Thus, thedominating proteins in enamel matrix, amelogenins, have been found toexist in several different sizes that together form supramolecularaggregates. They are markedly hydrophobic substances that underphysiologically conditions form aggregates. They may carry or becarriers for other proteins or peptides.

[0081] Other protein substances are also contemplated to be suitable foruse according to the present invention. Examples include proteins suchas proline-rich proteins and polyproline. Other examples of substancesthat are contemplated to be suitable for use according to the presentinvention are aggregates of such proteins, of enamel matrix derivativesand/or of enamel matrix proteins as well as metabolites of enamelmatrix, enamel matrix derivatives and enamel matrix proteins. Themetabolites may be of any size ranging from the size of proteins to thatof short peptides.

[0082] As mentioned above, the proteins, polypeptides or peptides foruse according to the invention typically have a molecular weight of atthe most about 120 kDa such as, e.g., at the most 100 kDa, 90 kDa, 80kDa, 70 kDa or 60 kDa as determined by SDS PAGE electrophoresis.

[0083] The proteins for use according to the invention are normallypresented in the form of a preparation, wherein the protein content ofthe active enamel substance in the preparation is in a range of fromabout 0.05% w/w to 100% w/w such as, e.g., about 5-99% w/w, about 10-95%w/w, about 15-90% w/w, about 20-90% w/w, about 30-90% w/w, about 40-85%w/w, about 50-80% w/w, about 60-70% w/w, about 70-90% w/w, or about8090% w/w.

[0084] A preparation of an active enamel substance for use according tothe invention may also contain a mixture of active enamel substanceswith different molecular weights.

[0085] The proteins of an enamel matrix can be divided into a highmolecular weight part and a low molecular weight part, and it has beenfound that a well-defined fraction of enamel matrix proteins possessesvaluable properties with respect to treatment of periodontal defects(i.e. periodontal wounds). This fraction contains acetic acidextractable proteins generally referred to as amelogenins andconstitutes the low molecular weight part of an enamel matrix (cf.EP-B-0 337 967 and EP-B-0 263 086).

[0086] The low molecular weight part of an enamel matrix has a suitableactivity for inducing binding between hard tissues in periodontaldefects. In the present context, however, the active proteins are notrestricted to the low molecular weight part of an enamel matrix. Atpresent, preferred proteins include enamel matrix proteins such asamelogenin, tuftelin, etc. with molecular weights (as measured in vitrowith SDS-PAGE) below about 60,000 Dalton but proteins having a molecularweight above 60,000 Dalton have also promising properties as candidatesfor promoting connective tissue growth.

[0087] Accordingly, it is contemplated that the active enamel substancefor use according to the invention has a molecular weight of up to about40,000 such as, e.g. a molecular weight of between about 5,000 and about25,000.

[0088] By separating the proteins, e.g. by precipitation, ion-exchangechromatography, preparative electrophoresis, gel permeationchromatography, reversed phase chromatography or affinitychromatography, the different molecular weight amelogenins can bepurified.

[0089] The combination of molecular weight amelogenins may be varied,from a dominating 20 kDa compound to an aggregate of amelogenins withmany different molecular weights between 40 and 5 kDa, and to adominating 5 kDa compound. Other enamel matrix proteins such as tuftelinor proteolytic enzymes normally found in enamel matrix can be added andcarried by the amelogenin aggregate.

[0090] In general, the enamel matrix, enamel matrix derivatives andenamel matrix proteins are hydrophobic substances, i.e. less soluble inwater, especially at increased temperatures. In general, these proteinsare soluble at non-physiological pH values and at a low temperature suchas about 4-20° C., while they will aggregate and precipitate at bodytemperature (35-37° C.) and neutral pH.

[0091] The enamel matrix, enamel matrix derivatives and/or enamel matrixproteins for use according to the invention comprise an active enamelsubstance, wherein at least a part is in the form of aggregates or afterapplication in vivo is capable of forming aggregates. The particle sizeof the aggregates is in a range of from about 20 nm to about 1 μm.

[0092] In accordance to the present invention, an active enamelsubstance may be used together with other active drug substances suchas, e.g. anti-bacterial, anti-inflammatory, antiviral, antifungalsubstances or in combination with local chemotherapy, inducers ofapoptosis, growth factors such as, e.g., TGFβ, PDGF, IGF, FGF, EGF,keratinocyte growth factor or peptide analogues thereof. Enzymes—eitherinherently present in the enamel matrix or preparation thereof oradded—may also be used in combination with an enamel matrix, enamelmatrix derivative and/or enamel matrix protein, especially proteases.

[0093] A preparation of an active enamel substance is normallyformulated as a pharmaceutical or cosmetic composition. Such acomposition may of course consist of the proteinaceous preparation or itmay further comprise a pharmaceutically or cosmetically acceptableexcipient. Especially suitable excipients for use in pharmaceutic orcosmetic compositions are propylene glycol alginate, or hyaluronic acidor salts or derivatives thereof.

[0094] In the following, examples of suitable compositions containing anactive enamel substance(s) are given. Depending on the use of the activeenamel substance(s), a composition may be a pharmaceutical or a cosmeticcomposition. In the following the term “pharmaceutical composition” isalso intended to embrace cosmetic compositions as well as compositionsbelonging to the so-called grey area between pharmaceuticals andcosmetics, namely cosmeceuticals.

[0095] For the administration to an individual (an animal or a human),an active enamel substances and/or a preparation thereof is preferablyformulated into a pharmaceutical composition containing the activeenamel substance and, optionally, one or more pharmaceuticallyacceptable excipients.

[0096] A composition comprising the active enamel substance to beadministered may be adapted for administration by any suitable route,e.g. by topical administration through a syringe, or by administrationto a tissue cavity through a hose or draining device. Furthermore, acomposition may be adapted to administration in connection with surgery,e.g. in connection with incision within the body in order to promoterepair and filling of deep cavity-shaped wounds, substantial tissuelosses after surgery and tissue defects.

[0097] As mentioned above, a composition of the active enamelsubstance(s) may be suitable for use during surgery, e.g. for localapplication (e.g. in a breast or in the abdominal wall) in the form of agel, film or dry pellet to induce fibroblast invasion, proliferation andgrowth.

[0098] The compositions may be formulated according to conventionalpharmaceutical practice, see, e.g., “Remington's PharmaceuticalSciences” and “Encyclopedia of Pharmaceutical Technology”, edited bySwarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.

[0099] As mentioned above, the application of a composition comprisingan active enamel substance is intended for inducing and stimulatingconnective tissue cell invasion, proliferation and growth as part of arepair process following significant removal or loss of tissue. Otherapplications may of course also be relevant such as application directlyin or into a deep wound or other substantial tissue defects.

[0100] A pharmaceutical composition comprising an active enamelsubstance serves as a drug delivery system. In the present context theterm “drug delivery system” denotes a pharmaceutical composition (apharmaceutical formulation or a dosage form) that upon administrationpresents the active substance to the body of a human or an animal. Thus,the term “drug delivery system” embraces plain pharmaceuticalcompositions such as, e.g., creams, ointments, liquids, powders,tablets, etc. as well as more sophisticated formulations such asdressings, devices, templates, smart-gels, grafts etc.

[0101] Apart from the active enamel substance, a pharmaceuticalcomposition for use according to the invention may comprisepharmaceutically or cosmetically acceptable excipients.

[0102] A pharmaceutically or cosmetically acceptable excipient is asubstance that is substantially harmless to the individual to which thecomposition is to be administered. Such an excipient normally fulfilsthe requirements given by the national health authorities. Officialpharmacopoeias such as e.g. the British Pharmacopoeia, the United Statesof America Pharmacopoeia and The European Pharmacopoeia set standardsfor pharmaceutically acceptable excipients.

[0103] Whether a pharmaceutically acceptable excipient is suitable foruse in a pharmaceutical composition is generally dependent on which kindof dosage form is chosen for use for a particular kind of wound. In thefollowing examples of suitable pharmaceutically acceptable excipientsare given for use in different kinds of compositions for use accordingto the invention.

[0104] In the following is given a review on relevant pharmaceuticalcompositions for use according to the invention. The review is based onthe particular route of administration. However, it is appreciated thatin those cases where a pharmaceutically acceptable excipient may beemployed in different dosage forms or compositions, the application of aparticular pharmaceutically acceptable excipient is not limited to aparticular dosage form or of a particular function of the excipient.

[0105] The choice of pharmaceutically acceptable excipient(s) in acomposition for use according the invention and the optimumconcentration thereof cannot generally be predicted and must bedetermined on the basis of an experimental evaluation of the finalcomposition. However, a person skilled in the art of pharmaceuticalformulation can find guidance in e.g., “Remington's PharmaceuticalSciences”, 18th Edition, Mack Publishing Company, Easton, 1990.

[0106] For application into the tissue defect, the compositions for useaccording to the invention may contain conventionally non-toxicpharmaceutically acceptable carriers and excipients includingmicrospheres and liposomes.

[0107] The compositions for use according to the invention include allkinds of solid, semi-solid and fluid compositions. Compositions ofparticular relevance are e.g. pastes, ointments, hydrophilic ointments,creams, gels, hydrogels, solutions, emulsions, suspensions, powders,films, foams, pads, sponges (e.g. collagen sponges) and transdermaldelivery systems.

[0108] The pharmaceutically acceptable excipients may include solvents,buffering agents, preservatives, humectants, chelating agents,antioxidants, stabilizers, emulsifying agents, suspending agents,gel-forming agents, ointment bases, penetration enhancers, perfumes, andskin protective agents.

[0109] Examples of solvents are e.g. water, alcohols, vegetable ormarine oils (e.g. edible oils like almond oil, castor oil, cacao butter,coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil,peanut oil, poppy seed oil, rape seed oil, sesame oil, soybean oil,sunflower oil, and tea seed oil), mineral oils, fatty oils, liquidparaffin, polyethylene glycols, propylene glycols, glycerol, liquidpolyalkylsiloxanes, and mixtures thereof.

[0110] Examples of buffering agents are e.g. citric acid, acetic acid,tartaric acid, lactic acid, hydrogen phosphoric acid, diethylamine etc.

[0111] Suitable examples of preservatives for use in compositions areparabens, such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben,isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid,benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDMhydantoin, iodopropynyl butylcarbamate, EDTA, benzalconium chloride, andbenzylalcohol, or mixtures of preservatives.

[0112] Examples of humectants are glycerin, propylene glycol, sorbitol,lactic acid, urea, and mixtures thereof.

[0113] Examples of chelating agents are sodium EDTA and citric acid.

[0114] Examples of antioxidants are butylated hydroxy anisole (BHA),ascorbic acid and derivatives thereof, tocopherol and derivativesthereof, cysteine, and mixtures thereof.

[0115] Examples of emulsifying agents are naturally occurring gums, e.g.gum acacia or gum tragacanth; naturally occurring phosphatides, e.g.soybean lecithin; sorbitan monooleate derivatives; wool fats; woolalcohols; sorbitan esters; monoglycerides; fatty alcohols; fatty acidesters (e.g. triglycerides of fatty acids); and mixtures thereof.

[0116] Examples of suspending agents are e.g. celluloses and cellulosederivatives such as, e.g., carboxymethyl cellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum,tragacanth, and mixtures thereof.

[0117] Examples of gel bases, viscosity-increasing agents or componentswhich are able to take up exudate from a wound are: liquid paraffin,polyethylene, fatty oils, colloidal silica or aluminium, zinc soaps,glycerol, propylene glycol, tragacanth, carboxyvinyl polymers,magnesium-aluminium silicates, Carbopol®, hydrophilic polymers such as,e.g. starch or cellulose derivatives such as, e.g.,carboxymethylcellulose, hydroxyethylcellulose and other cellulosederivatives, water-swellable hydrocolloids, carragenans, hyaluronates(e.g. hyaluronate gel optionally containing sodium chloride), andalginates including propylene glycol aginate.

[0118] Examples of hydrophobic or water-emulsifying ointment bases areparaffins, vegetable oils, animal fats, synthetic glycerides, waxes,lanolin, and liquid polyalkylsiloxanes.

[0119] Examples of hydrophilic ointment bases are solid macrogols(polyethylene glycols).

[0120] Examples of powder components are: alginate, collagen, lactose,powder which is able to form a gel when applied to a wound (absorbsliquid/wound exudate). Normally, powders intended for application onlarge open wounds must be sterile and the particles present must bemicronized.

[0121] Examples of other excipients are polymers such as carmelose,sodium carmelose, hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acaciagum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates,cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates andkitosans.

[0122] The compositions mentioned above for topical administration aremost suitably for application directly into tissue defects or by anyconvenient route of administration.

[0123] Compositions which have proved to be of importance in connectionwith topical application are those which have tixothropic properties,i.e. the viscosity of the composition is affected e.g. by shaking orstirring so that the viscosity of the composition at the time ofadministration can be reduced and when the composition has been applied,the viscosity increases so that the composition remains at theapplication site.

[0124] Suitable compositions for use according to the invention may alsobe presented in the form of suspensions, emulsions or dispersions. Suchcompositions contain the active enamel substance in admixture with adispersing or wetting agent, suspending agent, and/or one or morepreservatives and other pharmaceutically acceptable excipients. Suchcompositions may also be suitable for use in the delivery of the activeenamel substance to e.g. an intact or damaged tissue with a cavity likedefect.

[0125] Suitable dispersing or wetting agents are, e.g., naturallyoccurring phosphatides, e.g., lecithin, or soybean lecithin;condensation products of ethylene oxide with e.g. a fatty acid, a longchain aliphatic alcohol, or a partial ester derived from fatty acids anda hexitol or a hexitol anhydride, e.g. polyoxyethylene stearate,polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitanmonooleate, etc.

[0126] Suitable suspending agents are, e.g., naturally occurring gumssuch as, e.g., gum acacia, xanthan gum, or gum tragacanth; cellulosessuch as, e.g., sodium carboxymethylcellulose, microcrystalline cellulose(e.g. Avicel® RC 591, methylcellulose); alginates and kitosans such as,e.g., sodium alginate, etc.

[0127] Suitable examples of preservatives for use in compositionsaccording to the invention are the same as those mentioned above.

[0128] In a pharmaceutical composition for use according to theinvention on skin or mucosa, an active enamel substance is generallypresent in a concentration ranging from about 0.01% to about 99.9% w/w.The amount of composition applied will normally result in an amount oftotal protein per cm² wound/tissue-defect area, corresponding to fromabout 0.01 mg/cm² to about 20 mg/cm² such as from about 0.1 mg/cm² toabout 15 mg/cm².

[0129] The amount applied of the composition depends on theconcentration of the active enamel substance in the composition and ofthe release rate of the active enamel substance from the composition,but is generally in a range corresponding to at the most about 15-20mg/cm².

[0130] In those cases where the active enamel substance is administeredin the form of a fluid composition, the concentration of the activeenamel substance in the composition is in a range corresponding to fromabout 0.1 to about 50 mg/ml. Higher formulation/dosages are in somecases desirable and can also be obtained such as of at least about 50mg/ml.

[0131] The concentration of the active enamel substance in apharmaceutical composition depends on the specific enamel substance, itspotency, the severity of the tissue loss or defect to be prevented ortreated, and the age and condition of the patient. Methods applicable toselecting relevant concentrations of the active enamel substance in thepharmaceutical composition are well known to a person skilled in the artand may be performed according to established guidelines for goodclinical practice (GCP) or Investigational New Drug Exemption (“IND”)regulations as described in e.g. International Standard ISO/DIS 14155Clinical investigation of medical devices, 1994 and ICH (InternationalCommittee for Harmonisation): Harmonised tripartite guideline for goodclinical practice, Brookwood Medical Publications, Ltd, Surrey, UK,1996. A person skilled in the art would, by use of the methods describedin standard textbooks, guidelines and regulations as described above aswell as common general knowledge within the field, be able to select theexact dosage regimen to be implemented for any active enamel substanceand/or selected other active substances and dosage form using merelyroutine experimentation procedures.

[0132] As will be understood, details and particulars concerning the useof an active enamel substance for the induction and stimulation ofconnective tissue cell invasion, proliferation and growth will be thesame as or analogous to the details and particulars concerning the otheruse aspects (anti scarring and contraction resistance) and the methodaspects discussed above, and this means that wherever appropriate, thestatements above concerning an active enamel substance, a preparationcontaining an active enamel substance, a pharmaceutical compositioncontaining an active enamel substance, preparation of i) an activeenamel substance, ii) a preparation containing an active enamelsubstance, iii) a pharmaceutical composition containing an active enamelsubstance, as well as improved properties and uses apply mutatismutandis to all aspects of the invention.

[0133] The observation, that enamel matrix is formed and temporarilypresent during root and root cementum formation can explain howapplication of enamel matrix, enamel matrix derivatives and/or enamelmatrix proteins promotes the regeneration of periodontal tissue.

[0134] However, the observation underlying the present invention thatenamel matrix, enamel matrix derivatives and/or enamel matrix proteinsalso have a positive effect on invasion, proliferation and growth ofconnective tissue cells is very surprising. The same applies to theobservations with respect to the reduced scarring and contractionobserved in treated defects. As demonstrated in the experimental sectionherein, the active enamel substance aggregates and revitalises radiatedfibroblasts cells and initiates fibroblast cell migration, replicationand growth.

EXPERIMENTS

[0135] The therapeutic and/or prophylactic activity of active enamelsubstances may of course be evidenced by in vivo tests, usingexperimental animals or humans. However, an indication of the efficacyand/or activity of enamel matrix, enamel matrix derivatives and/orenamel matrix proteins can be obtained by performing relatively simplein vitro tests such as, e.g., tests involving cell cultures.

[0136] Furthermore, there are several parameters that may be employed inorder to evaluate a wound healing effect. These include:

[0137] Ultra-sound analysis of treated tissues

[0138] Magnet resonance imaging (MR)

[0139] Histopathology/cytology (microscopic evaluation of wound tissuesand fluids)

[0140] Scintigraphy (radionuclide imaging of wound tissue)

Example 1

[0141] Investigation of Growth Behaviour of Dermal Fibroblasts Culturedin the Presence of Enamel Matrix Derivative.

[0142] The purpose of this example is to show the capacity of enamelmatrix derivative to induce and stimulate dermal fibroblast cellattachment, replication and growth.

[0143] Cell Isolation and Culture Conditions

[0144] Human dermal fibroblasts, obtained from ATTC, were cultured fromhealthy dermal tissues from young healthy volunteering individuals.Cultures were maintained in DMEM with 10% foetal bovine serum. EMD,EMDOGAIN® (BIORA AB, Sweden), was added prior to commencement of cellcultures, by coating charged plastic culture dishes with a 0.5 mg/ml EMDsolution in 0.1% HAc in PBS overnight. In addition, the medium wassupplemented with 100 μg EMD per millilitre. The EMD concentrations werechosen on the basis of pilot experiments with cultured cells indicatingthat optimal growth occurred at these values, and that cell growth didnot benefit from further addition of EMD. There were no changes of mediaduring the five to seven day observation period of this study. Allexperiments commenced with 50,000 cells per millilitre of culturemedium.

[0145] Cell Attachment

[0146] To assess the cell attachment rate during the first four hoursafter seeding, 100,000 cells were cultured on EMD coated surfaces for30, 60, 120 or 240 minutes before the cultures were vigorously washedwith PBS to remove all unattached cells. The washing solution wascentrifuged and the numbers of unattached cells were analysed using aBürker chamber. The attached cells were then removed from the surface bytrypsinisation and counted in the same way for control. Uncoated disheswere used as negative control.

[0147] Cell Culture Densities

[0148] Cells were seeded and maintained in cultures with or without EMDfor 24, 48, 72, 96 or 120 hours. Cultures were then carefully washedwith PBS and the number of attached cells per square millimetre wascalculated in the microscope using a fixed grid.

[0149] Cell Metabolism

[0150] Cells were cultured for 24, 48, 72, 96, 120 or 144 hours and thengiven a 4-hour pulse of 50 μCi [³⁵S]methionine (Cell culture grade,Amersham Pharmacia Biotech). The cultures were then washed with PBS andthe cells were removed by trypsinisation. The cells were then washedagain, centrifuged and 200 μl of each cell pellet were dissolved inUniverSol™ liquid scintillation cocktail (ICN Biomedicals Inc.) andcounted two times 300 seconds in a Packard Tricarb scintillationcounter.

[0151] Nucleic Acid Synthesis

[0152] Nucleic acid synthesis in cells cultured 24, 48, 72 or 96 hourswas assessed by colorimetric analysis at 370 nm following a 4-hour pulsewith BrdU using the Boehringer Mannheim Cell Proliferation ELISA, BrdUkit (Cat. No. 1647229). During the pulse, the pyrimidine analogue BrdUwas incorporated in place of thymidine into the newly synthesised DNA ofproliferating cells. At the end of the pulse the cells were washed,fixed and denatured and the amount of incorporated BrdU was measured byELISA utilising an anti-BrdU peroxidase conjugated antibody.

[0153] Results

[0154] The experiments showed that fibroblast cell attachment rateduring the first hours after seeding is nearly five times more efficientwhen the surface of the culture dish is coated with EMD (FIG. 1). Celldensity in the cultures increased faster when EMD was present (FIG. 2).The general trend was that cultures growing in the presence of EMD got aone-day lead, reaching confluence after four days in culture, one dayahead of the control cells. The metabolic rate of fibroblast cells alsoincreased on a per cell basis in cultures seeded on EMD as compared tocontrol cultures (FIG. 3). This increase in [³⁵S]methionine utilisationwas higher than the observed change in growth rate alone.

[0155] In pulse-chase experiments with the thymidine analoguebromodeoxyuridine (BrdU) fibroblast cell cultures showed an increasedDNA synthesis in the presence of EMD (FIG. 4). The results prove thatEMD stimulates fibroblast proliferation and differentiation. Thefibroblasts in the experiment attached to the dishes and grew quickerthan those in the uncoated dishes, their metabolism was enhanced andthey most probably expressed cell-specific proteins.

Example 2

[0156] Investigation of Recovery of Fibroblast Cells Exposed to IonisingRadiation by Application of Enamel Matrix Derivative.

[0157] The purpose of this example is to show the ability of enamelmatrix derivative to revitalise fibroblast cells that have been exposedto ionising radiation.

[0158] Cell Isolation and Culture Conditions

[0159] Human dermal fibroblasts, obtained from ATTC, were cultured fromhealthy dermal tissues from young healthy volunteering individuals.Cultures were maintained in DMEM with 10% foetal bovine serum. EMD wasadded prior to commencement of cell cultures, by coating charged plasticculture dishes with a 0.5 mg/ml EMD solution in 0.10/% HAc in PBSovernight. In addition, the medium was supplemented with 100 μg of EMDper millilitre. The EMD concentrations were chosen on the basis of pilotexperiments with cultured cells indicating that optimal growth occurredat these values, and that cell growth did not benefit from furtheraddition of EMD. There were no changes of media during the five to sevenday observation period of this study. All experiments commenced with50,000 cells per millilitre of culture medium.

[0160] Radiation Therapy

[0161] Confluent cultures of dermal fibroblast cells, cultured withoutEMD, received a gamma radiation dosage of 0, 1, 2, 5 10, 15 and 20 Grey.Immediately after radiation therapy, the cultures were split in two bytrypsination and seeding according to standard operating procedures. Oneof the parallels was then cultured with 100 μg/ml EMD present in themedium, whereas the other was cultured without EMD as unstimulatedcontrol.

[0162] Cell Culture Densities

[0163] Cells were seeded and maintained in cultures with or without EMDfor 24, 48, 72, 96 or 120 hours. Cultures were then carefully washedwith PBS and the number of attached cells per square millimetre wascalculated in the microscope using a fixed grid.

[0164] Results

[0165] Radiated cells growing in the presence of EMD increased theirnumber twice as fast as unstimulated cultures (FIGS. 5A and 5B). Thistrend was true for all cultures, even though in general the growthpotential of the cultured dermal fibroblasts decreased with increasingradiation dosages. The results demonstrate that EMD can revitaliseradiated cultured dermal fibroblast cells, and that the presence of EMDproteins stimulates proliferation and growth of these cells also whenbasic cellular functions are impaired by radiation damage.

Example 3

[0166] Investigation of Soft Tissue Defect Fill and Prevention ofScarring and Tissue Contraction after Surgical Removal of a BreastTumour Followed by Radiation Therapy.

[0167] The purpose of this example is to show the influence of activeenamel substances on improvement of soft tissue defect fill and reducedtissue contraction after cytoreductive surgery and radiation therapy.

[0168] An active enamel substance may be applied either directly into asoft tissue defect prior to suturing or it may be injected into thewound cavity after suturing. The volume/amount of active enamelsubstances applied will differ from case to case and tissue to tissue,but mostly the therapy will aim at replacing the volume of the losttissue. However, in cases where an increase or decrease of the volume ofa tissue or body part, e.g. a breast, is desirable, the enamel matrix,enamel matrix derivatives and/or enamel matrix proteins may be appliedin surplus or deficit to acquire the desired outcome. The active enamelsubstance may be used as such or may be used in a suitable preparationor pharmaceutical composition.

[0169] A patient with a diagnosed adenocarcinoma in the breast issubmitted for surgical removal of the tumor according to standardaesthetic and surgical procedures. After resection of the tumor tissue,the mass of the removed tissue is estimated to about 5 ml. The woundcavity in the breast is then half closed by suturing at the incision,carefully avoiding pull, tension or contraction in the walls of thewound cavity. The wound cavity is then thoroughly rinsed with sterilesaline to remove blood clots, cell debris and damaged tissue. Afterthoroughly draining the saline from the wound cavity, EMD in PGA in theform of EMDOGAIN® Gel (BIORA AB, Sweden) is injected into the woundcavity so that the whole of the cavity is filled (volume>5 ml). Finally,the wound cavity is closed by sutures at the incision site, taking carethat the EMDOGAIN® Gel (BIORA AB, Sweden) remains in situ. No drainingdevice is applied, and standard postoperative procedures and wound careare applied.

[0170] The patient is allowed to recover from surgery for two weeksbefore he/she is submitted to adjuvant radiation therapy of the treatedbreast. Prior to the radiation therapy, the wound healing is assessed byclinical examination, ultrasound and imaging in order to monitorconnective tissue growth in the tissue defect. Clinical pictures andmeasurements of the size and location of the healing defect arerecorded.

[0171] To ensure total eradication of malignant cells from the breast,the patient is submitted to local radiation therapy. The radiationdosage depends on the phenotype of the malignant cells (assessed byhistology on tissue removed during surgery), the size of the breast andthe condition of the patient (age, weight, systemic diseases etc.). Thetypical dosage ranges between 0.5 and 20 Gray. If considered necessary,radiation therapy is repeated until the patient is declared free fromprimary tumour cells.

[0172] Following the first radiation therapy, the patient is monitoredevery week the first four weeks and then every month the next sixmonths, for connective tissue wound fill, tissue contractions andappearance. The healing process and cell invasion into the tissue defectis monitored by clinical measurements, palpation, photography, and/orultra sound imaging. The patient is asked to fill in a questionnaireregarding postoperative discomfort and the progression of the healing.If possible, aspiration biopsies are obtained from the healing breast toassess the type and quantity of cellular ingrowth in the defect.

[0173] In the frequent normal cases without the use of EMD, the woundcavity starts to contract after the first radiation therapy. This is dueto the removal of proliferating fibroblasts from the wound by radiation,leaving a cell poor dense connective tissue that shrinks as the woundorganises. At six months, the wound contraction often is so severe thatthe patients prefer to have most of the breast removed and replaced bysilicon inlays. Thus, conservative surgery in breasts scheduled forradiation therapy often fails. However, by application of EMD into thewound cavity prior to radiation therapy, a wound fill comprising cellrich, loose connective tissue that is less sensitive to radiationinduced contraction is achieved. By reducing contraction in thesewounds, the post radiation wound healing is improved and the need foradditional corrective surgery and/or prosthesis treatment issignificantly reduced.

LIST OF REFERENCES

[0174] 1. Hammarström et al., 1997, Journal of Clinical Periodontology24, 658-668

[0175] 2. Lyngstadaas et al., 2000, Journal of Clinical Periodontology27, 1-8

[0176] 3. Ten Cate: Oral Histology, 1994;

[0177] 4. Robinson: Eur. J. Oral Science, January 1998, 106 Suppl.1:282-91

[0178] 5. Janson, J-C & Rydén, L. (Eds.), Protein purification, VCHPublishers 1989

[0179] 6. Fincham et al. in J. Struct. Biol. 1994 March-April; 112(2):103-9 and in 3. Struct. Biol. 1995 July-August; 115(1): 50-9)

[0180] 7. “Remington's Pharmaceutical Sciences” and “Encyclopedia ofPharmaceutical Technology”, edited by Swarbrick, J. & J. C. Boylan,Marcel Dekker, Inc., New York, 1988

[0181] 8. “Remington's Pharmaceutical Sciences”, 18th Edition, MackPublishing Company, Easton, 1990

[0182] 9. International Standard ISO/DIS 14155 Clinical investigation ofmedical devices, 1994

[0183] 10. Harmonised tripartite guideline for good clinical practice,Brookwood Medical Publications, Ltd, Surrey, UK, 1996

[0184] 11. Harris, ELV & Angal, S., Protein purification methods—Apractical approach, IRL Press, Oxford 1990

[0185] 12. Sambrook, J. et al.: Molecular Cloning, Cold Spring HarborLaboratory Press, 1989

[0186] 13. Gestrelius S, Lyngstadaas SP, Hammarstrøm L.Emdogain—periodontal regeneration based on biomimicry. Clin Oral Invest4:120-125, 2000

1. Use of an active enamel substance for the preparation of apharmaceutical or cosmetic composition for filling a wound cavity and/ortissue defect that is characterised by a substantial loss of tissue. 2.Use according to claim 1 for improving cellular re-fill of said woundcavity and/or tissue defect.
 3. Use according to claims 1 and 2, whereinsaid cellular re-fill is characterised as soft tissue filling.
 4. Useaccording to any one of claims 1-3, wherein said composition activelystimulates cellular neogenesis in said wound cavity and/or tissuedefect.
 5. Use according to any one of claims 1-4, wherein saidcomposition actively stimulates cellular proliferation, differentiationand/or maturation in said wound cavity and/or tissue defect.
 6. Useaccording to claim 4 or 5, wherein said active stimulation is cell-typespecific.
 7. Use according to claim 6, wherein said active stimulationis cell-type specific for a cell from mesodermal and/or endodermalorigin.
 8. Use according to any one of claims 1-7, wherein the cavity ordefect is caused by cytoreductive surgery.
 9. Use according to any oneof claims 1-8, wherein the cavity or defect is at least partly caused byradiation therapy.
 10. Use according to any one of claims 1-7, whereinthe cavity or defect is caused by a bodily injury, infection or atrauma.
 11. Use according to any one of claims 1-7, wherein said cavityor defect is caused by the surgical removal of a tumour selected fromthe group consisting of mammalian neoplasm, particularly neck and headcancer, abdominal cancer, ovarian cancer, breast cancer and skin cancer.12. Use according to claim 11, wherein said cavity or defect is causedby the surgical removal of a breast tumour.
 13. Use according to any oneof claims 1-12 for the prevention and/or treatment of scarring and woundcontraction.
 14. Use according to claim 13 for the prevention and/ortreatment of scarring and contraction in a tissue selected from thegroup consisting of breast tissue, lip tissue, abdominal wall tissue,facial tissue, neck tissue, tissue of the extremities, soft tissue ormuscle tissue.
 15. Use according to claim 14, wherein scarring andcontraction is prevented or treated that affects a normal function of abreast, lip, abdominal wall, face, neck and/or extremities.
 16. Useaccording to any one of claims 13-15, wherein scarring and contractionis prevented or treated that affects the appearance of the patient. 17.Use according to any one of claims 13-16, wherein the scarring andcontraction is at least partly caused by infection, malnutrition,inflammation, systemic diseases, pathological conditions, abnormalanatomical conditions and/or misconduct.
 18. Use according to any of thepreceding claims, wherein the active enamel substance is enamel matrix,enamel matrix derivatives and/or enamel matrix proteins.
 19. Useaccording to any of the preceding claims, wherein the active enamelsubstance is selected from the group consisting of enamelins,amelogenins, non-amelogenins, proline-rich non-amelogenins, tuftelins,and derivatives thereof and mixtures thereof.
 20. Use according to anyof the preceding claims, wherein the active enamel substance has amolecular weight of at the most about 120 kDa such as, e.g., at the most100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS PAGEelectrophoresis.
 21. Use according to any of the preceding claims,wherein the preparation of an active enamel substance contains a mixtureof active enamel substances with different molecular weights.
 22. Useaccording to any of the preceding claims, wherein the preparation of anactive enamel substance comprises at least two substances selected fromthe group consisting of amelogenins, proline-rich non-amelogenins,tuftelin, tuft proteins, serum proteins, salivary proteins,ameloblastin, sheathlin, and derivatives thereof.
 23. Use according toany of the preceding claims, wherein the active enamel substance has amolecular weight of up to about 40,000.
 24. Use according to any of thepreceding claims, wherein the active enamel substance has a molecularweight of between about 5,000 and about 25,000.
 25. Use according to anyof the preceding claims, wherein the major part of the active enamelsubstance has a molecular weight of about 20 kDa.
 26. Use according toany of the preceding claims, wherein at least a part of the activeenamel substance is in the form of aggregates or after application invivo is capable of forming aggregates.
 27. Use according to claim 26,wherein the aggregates have a particle size of from about 20 nm to about1 μm.
 28. Use according to any of the preceding claims, wherein theprotein content of the active enamel substance in the preparation is ina range of from about 0.05% w/w to 100% w/w such as, e.g., about 5-99%w/w, about 10-95% w/w, about 15-90% w/w, about 20-90% w/w, about 30-90%w/w, about 40-85% w/w, about 50-80% w/w, about 60-70% w/w, about 70-90%w/w, or about 80-90% w/w.
 29. Use according to any of the precedingclaims, wherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient.
 30. Use according to claim 29,wherein the pharmaceutically acceptable excipient is propylene glycolalginate.
 31. Use according to claim 29, wherein the pharmaceuticalcomposition comprises 30 mg of Enamel Matrix protein and 1 ml of vehiclesolution (Propylene Glycol Alginate).
 32. Method for promoting thefilling of a wound cavity and/or tissue defect in a patient, said cavityand/or deffect being characterised by a substantial loss of tissue,wherein a pharmaceutical composition comprising an active enamelsubstance is administered to said wound cavity and/or tissue defect. 33.Method for promoting the re-filling of a wound cavity and/or tissuedefect in a patient with soft tissue filling, said cavity and/or deffectbeing characterised by a substantial loss of tissue, wherein apharmaceutical composition comprising an active enamel substance isadministered to said wound cavity and/or tissue defect.
 34. Methodaccording to any of claims 32 or 33, wherein said administeredcomposition actively stimulates cellular neogenesis of cells withmesodermal and/or endodermal origin in said wound cavity and/or tissuedefect.
 35. Method according to any of claims 32-34, wherein the cavityor defect is caused by cytoreductive surgery and/or wherein the cavityor defect is at least partly caused by radiation therapy.
 36. Methodaccording to claim 35, wherein said cavity or defect Is caused by thesurgical removal of a tumour selected from the group consisting ofmammalian neoplasm, particularly neck and head cancer, abdominal cancer,ovarian cancer, breast cancer and skin cancer.